Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The prevalence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis has been increasing to alarming levels globally. This has been exacerbated by tuberculosis (TB) co-infection with HIV where the epidemic is endemic. South Africa as a developing country is hit hard by TB and efforts to develop TB drugs that are compatible with anti-retroviral medication and also effective against MDR/XDR, could help shorten the treatment duration of the current TB treatment regimens. This thesis presents the identification and characterisation of a novel furanone based compound (F1082) and its derivatives as leads for anti-TB drug development. Furanones are generally known for an array of biological activities ranging from antibacterial, antifungal and antitumor. F1082 has an aromatic benzene structure and was identified from screening synthetic compounds against M. tuberculosis. It is potent against M. tuberculosis at minimum inhibitory concentration (MIC) of 8 μg/ml. It is selective for mycobacteria since it did not inhibit the growth of Gram-positive and Gram-negative bacteria at concentrations five times the MIC for M. tuberculosis. F1082 is generally bacteriostatic around MIC concentrations in its effects against M. tuberculosis however; it may be bactericidal at higher concentrations. It is as effective against MDR, XDR and clinical isolates of M. tuberculosis at the same concentration as the M. tuberculosis H37Rv reference strain. This suggests that F1082 may have a different mechanism of action compared to current TB drugs. It has been shown to have no antagonistic effect with the first-line anti-TB drugs and it has been shown to synergize with rifampicin by reducing the MIC of rifampicin. A drawback of F1082 is that it is cytotoxic to human cell lines, but this is presently being addressed through the synthesis of analogues that have shown improved activity and less cytotoxicity. The synthesis of more than 40 analogues has led to identification of 4 compounds that have more than five times higher activity and more than 100 times less cytotoxicity against human cell-lines. Microarray analyses have identified possible metabolic pathway/s in M. tuberculosis that is/are affected by F1082. One subset of genes which showed the most prominent alteration encodes the siderophores, which are involved with iron homeostasis in the M. tuberculosis bacillus. Of these genes, 7 were of interest (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH and bfrB) as they all fall in the same cluster and are involved in iron acquisition. Due to the involvement of iron we also show that F1082 generates oxidative stress that is metal (iron) dependent. From the results we conclude that F1082 is a promising antituberculosis lead compound with unique target properties and also specificity against mycobacteria. / AFRIKAANSE OPSOMMING: Die voorkoms van veelvuldige middelweerstandige M.tuberculosis (MDR) en uiters middelweerstandige M.tuberculosis (XDR) is besig om toe te neem teen ‘n kommerwekkende tempo wêreldwyd. Hierdie situasie word vererger met die ko-infektering van M.tuberculosis en HIV. Suid- Afrika, as ontwikkelende land, word sleg benadeel met tuberkulose siekte. Antituberkulose middels wat kan saamwerk met bestaande antiretrovirale middels en ook effektief is teen MDR en XDR stamme, kan alles meewerk om die behandelingstyd van tuberkulose te verkort. In hierdie tesis identifiseer en karakteriseer ons ‘n furanoon-gebaseerde verbinding (F1082) en derivate daarvan as voorloper-middels vir anti-tuberkulose middelontwikkeling. Furanone is algemeen bekend vir ‘n verskeidenheid van biologiese aktiwiteite insluitende antibakteriële-, antifungale- en antitumor aktiwiteite. F1082 bevat ‘n aromatiese benseenstruktuur en is oorspronklik geïdentifiseer gedurende die skandering van sintetiese middels teen M.tuberculosis. Dit het ‘n sterk werking teen M.tuberculosis met ‘n minimum inhibitoriese konsentrasie (MIC) van 8ug/ml. Dit is baie selektief vir mikobakterieë aangesien dit nie gram-positiewe of gram-negatiewe bakterieë teen 5 maal die MIC, soos vir M.tuberculosis, geïnhibeer het nie. F1082 is bevind om, by laer konsentrasies, bakteriostaties te wees in sy aktiwiteit teen M.tuberculosis maar by hoër konsentrasies word ‘n meer bakteriosidiese effek waargeneem. F1082 is effektief teen MDR, XDR en kliniese isolate van M.tuberculosis en teen dieselfde konsentrasie soos vir die M. tuberculosis H37Rv verwysingstam waargeneem is. Dit impliseer dat F1082 dalk ‘n alternatiewe meganisme van werking het in vergelyking met die van die huidige TB teenmiddels. F1082 toon geen antagonistiese werking in kombinasie met die voorste anti- TB middels nie, maar toon wel sinergistiese werking in kombinasie met rifampisien. F1082 toon nog sitotoksiese aktiwiteit teenoor menslike sellyne, maar die sintese van derivate van F1082 toon tot dusvêr groter anti-TB aktiwiteit en verminderde sitotoksisiteit. Die sintese van meer as 40 homoloë het gelei tot die identifisering van vier verbindings met vyf keer hoër anti-TB aktiwiteit en honderd keer verminderde sitotoksisiteit teen menslike sellyne as F1082 self. “Microarray” ontledings het ‘n aantal metabolise paaie geïdentifiseer waar F1082 ‘n effek kan uitoefen. Een stel gene wat die mees uitstaande effek toon kodeer vir siderofore wat betrokke is by yster homeostase in M.tuberculosis. Van hierdie gene was daar sewe van belang omdat hulle in dieselfde groep voorkom en almal betrokke is by ysteropname (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH, bfrB). Weens die rol wat F1082 in ysterhomeostase speel, toon ons ook dat F1082 intrasellulêre oksidatiewe stres bevorder wat yster afhanklik is. Al ons resultate dui daarop dat F1082 ‘n belowende ant-TB voorloper verbinding is met spesifisiteit teen M.tb en unieke teikeneienskappe in M. tuberculosis.

The current, grim world-TB scenario, with TB being the single largest infectious disease killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003). This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited. The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development. In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase. Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded. The availability of only three crystal structures for a critical enzyme like dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter. It is interesting to note that the P-loop-containing nucleoside monophosphate kinases (NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches. As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom. Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.

A tuberculose (TB) é uma doença causada pelo Mycobacterium tuberculosis. De acordo com estimativas da Organização Mundial da Saúde, a tuberculose é responsável pela morte de ~2 a 3 milhões de pessoas/ano no mundo e nos próximos 15 anos cerca de 1 bilhão de pessoas deverão ser infectadas, e destas, aproximadamente 35 milhões deverão morrer. Apesar de existirem vários medicamentos sendo utilizados no tratamento da doença, constatasse o crescimento no número de casos devido, principalmente, às variedades resistentes do M. tuberculosis. Considerando-se o aparecimento de cepas resistentes em TB, recomenda-se que novos medicamentos e/ou alvos biológicos alternativos devam ser intensivamente pesquisados. A ribonucleotídeo redutase (RNR), por exemplo, é uma proteína de interesse, pois catalisa uma etapa importante e única na síntese de novo dos dNTPs, reduzindo o ribonucleosídeo 5\' -difosfato ao seu correspondente desoxirribonucleosídeo 5\' -difosfato. A RNR é importante na síntese do DNA, e portanto, na divisão das células. Esta enzima importante, que possuí 16% de homologia com a RNR de mamíferos, é um alvo potencial para o desenvolvimento de novos fármacos, com provável aplicação no tratamento do câncer, da malária e do tripanossoma. Sabe-se que diferentes classes de compostos, através de diferentes mecanismos de ação, inibem a RNR, incluindo as α-(N)-heterocíclicas carboxaldeído tiossemicarbazonas, um dos inibidores mais potentes da RNR. Sabe-se que alguns derivados da tiossemicarbazona, inibidoras da RNR de células tumorais, apresentam atividade frente o M. tuberculosis atuando provavelmente através do mesmo mecanismo, envolvendo a inibição da correspondente RNR. Neste contexto, nesta tese de doutorado, foram aplicadas diferentes abordagens de QSAR/QSAR-3D no estudo de 40 derivados da 2-piridino-carboxaldeído tiossemicarbazona, inibidores da RNR de células H.Ep.-2, retirados de literatura selecionada (French & Blanz-Jr. 1974). Estes compostos foram divididos em cinco séries, a saber: séries A, B, C, D, e E contendo, respectivamente, 40, 39, 30, 23 e 22 compostos, na tentativa de tornar estas séries estruturalmente mais homogêneas. Para cada série, foram criados três grupos de treinamento e os respectivos grupos de teste (I, II e III), visando-se avaliar o poder de predição dos modelos gerados através das análises de QSAR/QSAR-3D. Para as análises de QSAR clássico, foram utilizados como variáveis independentes, os descritores mais relevantes gerados através do programa DRAGON e, pré-selecionados por PLS. Considerando-se a ausência de informações sobre a estrutura cristalográfica da enzima RNR do M. tuberculosis, os estudos de QSAR-3D foram iniciados empregando-se metodologias propostas em CoMFA e, em CoMSIA, implementadas no programa SYBYL. Além destas, foi realizada a modelagem por homologia da RNR do M. tuberculosis, utilizando-se o programa WHATIF. Para as abordagens CoMFA e CoMSIA as geometrias otimizadas através do método semi-empírico AM1 foram alinhadas átomo-a-átomo e, através da similaridade dos respectivos campos estéricos e eletrostáticos, utilizando-se o programa SEAL. Nos dois procedimentos a geometria do composto não substituído, um dos mais ativos na série, foi utilizada como molde considerando-se a ausência de informações sobre a conformação bioativa. A modelagem da RNR por homologia foi realizada utilizando-se como molde as estruturas cristalográficas, respectivamente, do C. ammoniagenes (código PDB 1KGN) e da S. typhimurium (código PDB 1R2F), sambas apresentando valores de identidade superior a 65%. Mais recentemente foram publicados os dados cristalográficos para a cadeia beta (subunidade menor) da RNR do M. tuberculosis (código PDB 1UZR). Os modelos CoMFA e CoMSIA gerados apresentaram valores aceitáveis para os coeficientes de correlação de predição, com altos valores para os coeficientes de correlação ajustados e baixos valores para os erros padrões. Os melhores modelos CoMFA e CoMSIA foram obtidos considerando o grupo com substituintes apenas na posição 5 do anel piridínico. Razoáveis coeficientes de correlação de predição para os modelos CoMSIA com altos coeficientes de correlação de ajuste e baixos valores para os erros padrões forma obtidos. Os mapas de contorno gerados em CoMFA e CoMSIA sugerem que grupos aceptores de ligações de hidrogênio próximos ao nitrogênio do anel piridínico deverá aumentar o valor da atividade inibitória. Esta observação está em boa concordância com os dados da literatura, na qual a formação de um complexo entre a TSC e o íon Ferro foi sugerido para a inibição da RNR. Estes estudos deverão permitir um melhor entendimento sobre as características estruturais desta classe de TSC inibidoras da RNR, como agentes antitumorais, em termos dos campos estéricos, eletrostáticos, hidrofóbico, doador e aceptor de ligações de hidrogênio, bem como a contribuição para o desenvolvimento racional de novos inibidores para esta importante enzima. Adicionalmente, dois compostos preparados em nosso laboratório, demonstraram atividade frente o M. tuberculosis, em testes realizados in vivo. / Tuberculosis is an illness caused by Mycobacterium tuberculosis. Data from World Health Organization (WHO) estimates, that about 2-3 millions of human population died by Mycobacterium tuberculosis infection and that during the next 15 years about 1 billion will be infected and 35 million will certainly die. Although, in the clinic it was found several antiTBdrugs, these numbers will increase due several reasons including M. tuberculosis resistant strains. It has been stressed the importance of novel medicines and/or alternative biological targets research projects. It is known that Ribonucleotide reductase (RNR), is an enzyme that catalyses the rate limiting step in the de novo synthesis of dNTPs, reducing the ribonucleoside 5\'-diphosphates to the corresponding deoxyribonuc1eoside 5\' -diphosphates. RNR has a critical role in the DNA synthesis and, hence, cell division. This key enzyme, that shows 16% homology when compared with mammals RNR, is a potential target for drug design of cell growth inhibitors, with potential application in cancer therapy, antimalaria and trypanosome chemotherapy. It is known that different types of compounds or species by means of different mechanism pathways can show RNR inhibition, including α-(N)-heterocyclic carboxaldehydes thiosemicarbazones that are one of the most potent classes of RNR inhibitors. More than that, some of them, that shows activity against M. tuberculosis seems to follow the same mechanism pathways proposed to the thiosemicarbazones tumor cells activity that means, that they probably are RNR inhibitors. In this study, a series of 40 α-(N)-2-formyl-pyridine thiosemicarbazone derivatives tested against RNR of H.ep.-2-cells (human epidermoid carcinoma), taken from selected literature (French & Blanz-Jr. 1974), has quantitatively analyzed by means of several QSAR/3D-QSAR approaches. These compounds were divided into 5 individual subsets, namely A, B, C, D, and E, having 40, 39, 30, 23 e 22 compounds, respectively. This procedure has been done in order to achieve more structurally homogeneous subsets. For each set, three individual training and test sets (I,II and III) have been created in order to evaluate the predictivity power of the generated QSAR/3D-QSAR models. QSAR analysis have been done using descriptors generated by DRAGON program that have been further pre-selected by PLS procedures. Considering that crystallographic data of RNR M. tuberculosis are not available in the literature, 3D-QSAR studies have been done these applying, initially, CoMFA and CoMSIA approaches, implemented in SYBYL. Homology model studies have been performed with WHATIF program CoMFA e CoMSIA approaches used optimized geometry obtained by semi-empirical AM1 methods that have been aligned by two different methods. Rigid alignment, in which the compounds were fitted atom-by-atom onto a template, based on the root mean square fit. The N(l) and C(2) atoms of the pyridine moiety and the heavy atoms of thiosemicarbazone backbone of TSC were used as template structure. (2) Field based, in which the steric and electrostatic fields, generated by the SEAL program were considered in the alignment. In both procedures the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation, has been taken as template. Homology RNR models were done using as template crystallographic data of ammoniagenes (1KGN) and S. typhimurium (1R2F) as template, respectively, with identity larger than 65%. More recent1y new crystallographic data have been published for the beta chain (smaller subunity) of RNR do M. tuberculosis (1UZR). CoMFA and CoMSIA generated models showed acceptable predictive correlation coefficients with high fitted correlation coefficients and low standard errors. Betler CoMFA and CoMSIA models have been derived considering a homogeneous subset of TSC substituted only at 5-position in pyridine ring. Reasonable predictive correlation coefficients for CoMSIA models with high fitted correlation coefficients and very low standard errors were obtained. The derived CoMFA and CoMSIA countour maps suggested that a hydrogen bond acceptor near the nitrogen pyridine ring could enhance inhibitory activity value. This observation is in good agreement with literature, in which a complex formation between TSC and iron ion has been suggested, to RNR inhibition. These studies are expected to enhance the understanding of the structural features of this class of TSC-RNR inhibitors as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen donor and acceptor fields as well as to contribute to rational design of inhibitors of this key enzyme. Additionally, two compounds that have been prepared by us showed activity against M. tuberculosis using in vivo test system.

A tuberculose (TB) é uma doença causada pelo Mycobacterium tuberculosis. De acordo com estimativas da Organização Mundial da Saúde, a tuberculose é responsável pela morte de ~2 a 3 milhões de pessoas/ano no mundo e nos próximos 15 anos cerca de 1 bilhão de pessoas deverão ser infectadas, e destas, aproximadamente 35 milhões deverão morrer. Apesar de existirem vários medicamentos sendo utilizados no tratamento da doença, constatasse o crescimento no número de casos devido, principalmente, às variedades resistentes do M. tuberculosis. Considerando-se o aparecimento de cepas resistentes em TB, recomenda-se que novos medicamentos e/ou alvos biológicos alternativos devam ser intensivamente pesquisados. A ribonucleotídeo redutase (RNR), por exemplo, é uma proteína de interesse, pois catalisa uma etapa importante e única na síntese de novo dos dNTPs, reduzindo o ribonucleosídeo 5\' -difosfato ao seu correspondente desoxirribonucleosídeo 5\' -difosfato. A RNR é importante na síntese do DNA, e portanto, na divisão das células. Esta enzima importante, que possuí 16% de homologia com a RNR de mamíferos, é um alvo potencial para o desenvolvimento de novos fármacos, com provável aplicação no tratamento do câncer, da malária e do tripanossoma. Sabe-se que diferentes classes de compostos, através de diferentes mecanismos de ação, inibem a RNR, incluindo as α-(N)-heterocíclicas carboxaldeído tiossemicarbazonas, um dos inibidores mais potentes da RNR. Sabe-se que alguns derivados da tiossemicarbazona, inibidoras da RNR de células tumorais, apresentam atividade frente o M. tuberculosis atuando provavelmente através do mesmo mecanismo, envolvendo a inibição da correspondente RNR. Neste contexto, nesta tese de doutorado, foram aplicadas diferentes abordagens de QSAR/QSAR-3D no estudo de 40 derivados da 2-piridino-carboxaldeído tiossemicarbazona, inibidores da RNR de células H.Ep.-2, retirados de literatura selecionada (French & Blanz-Jr. 1974). Estes compostos foram divididos em cinco séries, a saber: séries A, B, C, D, e E contendo, respectivamente, 40, 39, 30, 23 e 22 compostos, na tentativa de tornar estas séries estruturalmente mais homogêneas. Para cada série, foram criados três grupos de treinamento e os respectivos grupos de teste (I, II e III), visando-se avaliar o poder de predição dos modelos gerados através das análises de QSAR/QSAR-3D. Para as análises de QSAR clássico, foram utilizados como variáveis independentes, os descritores mais relevantes gerados através do programa DRAGON e, pré-selecionados por PLS. Considerando-se a ausência de informações sobre a estrutura cristalográfica da enzima RNR do M. tuberculosis, os estudos de QSAR-3D foram iniciados empregando-se metodologias propostas em CoMFA e, em CoMSIA, implementadas no programa SYBYL. Além destas, foi realizada a modelagem por homologia da RNR do M. tuberculosis, utilizando-se o programa WHATIF. Para as abordagens CoMFA e CoMSIA as geometrias otimizadas através do método semi-empírico AM1 foram alinhadas átomo-a-átomo e, através da similaridade dos respectivos campos estéricos e eletrostáticos, utilizando-se o programa SEAL. Nos dois procedimentos a geometria do composto não substituído, um dos mais ativos na série, foi utilizada como molde considerando-se a ausência de informações sobre a conformação bioativa. A modelagem da RNR por homologia foi realizada utilizando-se como molde as estruturas cristalográficas, respectivamente, do C. ammoniagenes (código PDB 1KGN) e da S. typhimurium (código PDB 1R2F), sambas apresentando valores de identidade superior a 65%. Mais recentemente foram publicados os dados cristalográficos para a cadeia beta (subunidade menor) da RNR do M. tuberculosis (código PDB 1UZR). Os modelos CoMFA e CoMSIA gerados apresentaram valores aceitáveis para os coeficientes de correlação de predição, com altos valores para os coeficientes de correlação ajustados e baixos valores para os erros padrões. Os melhores modelos CoMFA e CoMSIA foram obtidos considerando o grupo com substituintes apenas na posição 5 do anel piridínico. Razoáveis coeficientes de correlação de predição para os modelos CoMSIA com altos coeficientes de correlação de ajuste e baixos valores para os erros padrões forma obtidos. Os mapas de contorno gerados em CoMFA e CoMSIA sugerem que grupos aceptores de ligações de hidrogênio próximos ao nitrogênio do anel piridínico deverá aumentar o valor da atividade inibitória. Esta observação está em boa concordância com os dados da literatura, na qual a formação de um complexo entre a TSC e o íon Ferro foi sugerido para a inibição da RNR. Estes estudos deverão permitir um melhor entendimento sobre as características estruturais desta classe de TSC inibidoras da RNR, como agentes antitumorais, em termos dos campos estéricos, eletrostáticos, hidrofóbico, doador e aceptor de ligações de hidrogênio, bem como a contribuição para o desenvolvimento racional de novos inibidores para esta importante enzima. Adicionalmente, dois compostos preparados em nosso laboratório, demonstraram atividade frente o M. tuberculosis, em testes realizados in vivo. / Tuberculosis is an illness caused by Mycobacterium tuberculosis. Data from World Health Organization (WHO) estimates, that about 2-3 millions of human population died by Mycobacterium tuberculosis infection and that during the next 15 years about 1 billion will be infected and 35 million will certainly die. Although, in the clinic it was found several antiTBdrugs, these numbers will increase due several reasons including M. tuberculosis resistant strains. It has been stressed the importance of novel medicines and/or alternative biological targets research projects. It is known that Ribonucleotide reductase (RNR), is an enzyme that catalyses the rate limiting step in the de novo synthesis of dNTPs, reducing the ribonucleoside 5\'-diphosphates to the corresponding deoxyribonuc1eoside 5\' -diphosphates. RNR has a critical role in the DNA synthesis and, hence, cell division. This key enzyme, that shows 16% homology when compared with mammals RNR, is a potential target for drug design of cell growth inhibitors, with potential application in cancer therapy, antimalaria and trypanosome chemotherapy. It is known that different types of compounds or species by means of different mechanism pathways can show RNR inhibition, including α-(N)-heterocyclic carboxaldehydes thiosemicarbazones that are one of the most potent classes of RNR inhibitors. More than that, some of them, that shows activity against M. tuberculosis seems to follow the same mechanism pathways proposed to the thiosemicarbazones tumor cells activity that means, that they probably are RNR inhibitors. In this study, a series of 40 α-(N)-2-formyl-pyridine thiosemicarbazone derivatives tested against RNR of H.ep.-2-cells (human epidermoid carcinoma), taken from selected literature (French & Blanz-Jr. 1974), has quantitatively analyzed by means of several QSAR/3D-QSAR approaches. These compounds were divided into 5 individual subsets, namely A, B, C, D, and E, having 40, 39, 30, 23 e 22 compounds, respectively. This procedure has been done in order to achieve more structurally homogeneous subsets. For each set, three individual training and test sets (I,II and III) have been created in order to evaluate the predictivity power of the generated QSAR/3D-QSAR models. QSAR analysis have been done using descriptors generated by DRAGON program that have been further pre-selected by PLS procedures. Considering that crystallographic data of RNR M. tuberculosis are not available in the literature, 3D-QSAR studies have been done these applying, initially, CoMFA and CoMSIA approaches, implemented in SYBYL. Homology model studies have been performed with WHATIF program CoMFA e CoMSIA approaches used optimized geometry obtained by semi-empirical AM1 methods that have been aligned by two different methods. Rigid alignment, in which the compounds were fitted atom-by-atom onto a template, based on the root mean square fit. The N(l) and C(2) atoms of the pyridine moiety and the heavy atoms of thiosemicarbazone backbone of TSC were used as template structure. (2) Field based, in which the steric and electrostatic fields, generated by the SEAL program were considered in the alignment. In both procedures the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation, has been taken as template. Homology RNR models were done using as template crystallographic data of ammoniagenes (1KGN) and S. typhimurium (1R2F) as template, respectively, with identity larger than 65%. More recent1y new crystallographic data have been published for the beta chain (smaller subunity) of RNR do M. tuberculosis (1UZR). CoMFA and CoMSIA generated models showed acceptable predictive correlation coefficients with high fitted correlation coefficients and low standard errors. Betler CoMFA and CoMSIA models have been derived considering a homogeneous subset of TSC substituted only at 5-position in pyridine ring. Reasonable predictive correlation coefficients for CoMSIA models with high fitted correlation coefficients and very low standard errors were obtained. The derived CoMFA and CoMSIA countour maps suggested that a hydrogen bond acceptor near the nitrogen pyridine ring could enhance inhibitory activity value. This observation is in good agreement with literature, in which a complex formation between TSC and iron ion has been suggested, to RNR inhibition. These studies are expected to enhance the understanding of the structural features of this class of TSC-RNR inhibitors as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen donor and acceptor fields as well as to contribute to rational design of inhibitors of this key enzyme. Additionally, two compounds that have been prepared by us showed activity against M. tuberculosis using in vivo test system.